ABSTRACT Mapping the activity of an enzyme in vivo, directly within its natural environment, may provide unique insights into the role of a particular enzymes in pathophysiology of various diseases but remains extremely challenging. Recently, PI and colleagues demonstrated for the first time the ability of electron paramagnetic resonance (EPR) to image enzymatic activity using a nitroxide radical substrate of alkaline phosphatase (ALP). However, the in vivo application is hampered by the fast bioreducation of the nitroxide fragment. This project aims to develop the first biostable paramagnetic probes sensitive to enzymatic activity. The design is based on the triarylmethyl (TAM) radical scaffold known for its exceptional stability in biological milieu, narrow linewidth and high performance in polarization for Overhauser enhanced magnetic resonance imaging (OMRI). To achieve this goal we will rely on our recent advances in the chemistry of TAM functional probes that allow for multifunctional mapping using EPR imaging and/or OMRI. To prove the concept, under specific aim 1 (SA1) we will synthesize TAM-based paramagnetic substrates of alkaline phosphatase whose spectrum is modified upon enzymatic dephosphorylation. In addition, the synthesized probes will inherit the intrinsic sensitivity of TAM radical to tissue pO2, therefore making them dual function enzymatic ALP & pO2 probes. Under SA2 we will perform in vitro optimization of the OMRI sequences for dual ALP & pO2. Under SA3, as a first application of this new concept in vivo, we will image alkaline phosphatase (ALP) activity concurrently with tissue oxygenation using OMRI in the tumor microenvironment of a HeLa subcutaneous flank tumor model. Cancer research has recently experienced a paradigm shift from the seemingly obvious target of tumor cells towards key support systems of cancer, the tumor microenvironment (TME). Tissue pO2 and the activities of enzymes are among the important TME biomarkers. The imaging results will be validated using independent techniques, namely Oxylite for pO2 and optical imaging for ALP. The project focuses on the dual ALP & pO2 mapping using OMRI as a proof of concept but the probe scaffold has been designed for easy application to many other important enzymes in the future. The success of this proposal will provide a unique tool for in vivo enzymology allowing to map the activity of an enzyme and to correlate spacially this activity with tissue pO2. This unique tools will allows for outstanding applications in drug screening, therapy optimization, predication of the response to a particular treatment or to study the role of a particular enzyme in physiology and physiopathology.